This invention relates to focusing of samples, and more particularly to an automatic reverse focusing system for microscopic imaging instruments.
The diagnosis of physiological conditions such as cancer, infectious disease and prenatal disorders, is of paramount importance to the prevention, monitoring and treatment of such diseases. Typically, a biological specimen from a patient is used for analysis; identifying the presence of particular characteristic features of the disease indicates the presence or predisposition towards the disease. Biological sample analysis in its most traditional sense is performed by microscopic examination which, depending on the magnification, can be used to visualize the presence of abnormal numbers or types (e.g., cytology) of cells, organelles, organisms or biological markers.
Automated microscopic analysis systems have been developed to analyze specimens quickly and have the advantage of accuracy over manual analysis in which technicians may experience fatigue over time leading to inaccurate reading of the sample (see, e.g., the ACIS system, Chromavision, San Juan Capistrano, Calif.). Typically, samples on a slide are loaded onto the microscope, the microscope objective or lens focuses on a particular area of the sample, and the sample is scanned for particular features or objects of interest. To achieve an accurate image of the sample or particular features or objects in the sample, the sample must be focused. However, for various reasons present focusing methods and systems often have difficulty or require extended amounts of time to attain an optimal image focus. Improvement in image quality and the speed with which images are focused in microscopic systems will lead to improvements in sensitivity, speed, and accuracy of sample analysis and the diagnosis of disease by such systems. The invention mitigates this problem and provides related advantages.
One of the problems of current automated focusing systems is obtaining focused images. For automated focusing microscope systems, difficulties can be due to the presence of dust or dirt on the slide or coverslip, or the presence of imperfections or flaws, one or more of which can lead to an inability or a delay in obtaining a focused image. As shown in FIG. 1, dust particles may appear on top of the coverslip. As the objective travels from the top to the bottom, it may focus on the dust particles if the dust particles generate a strong signal of focus power. Although the sensitivity of the focus algorithm can be decreased in order not to focus on the dust, the disadvantage is that the focus algorithm may not be able to focus on the sample when the sample gives a weak focus power signal due, for example, to a light stain. The invention addresses such problems through the technique of reverse focusing. In this technique, the focus or focal plane of a lens, such as an objective of a microscope, is set so that the focal plane is located below a sample of which an image is to be acquired. After the focal plane is set to the desired position, an image is acquired at that position. After acquiring the first image, one or more subsequent images (second, third, fourth, fifth, etc.) are acquired after adjusting the position of the focal plane along the z-axis away from the first image focal plane, i.e., reverse focusing. In this way, the sample can be the first thing to be seen because when the stage or objective moves up, focusing on dust can be avoided. Thus, the focus algorithm can be set at a sensitivity to focus on samples that generate weak focus power signals.
The position of the focal plane can be set based upon knowledge of the positions of the objective or lens, the stage or other similar substrate supporting or positioning device, the thickness of the substrate (e.g., microscope slide) upon which a sample has been placed, and a coverslip, if used. In this way, the focal plane of an autofocusing system can be positioned at any location relative to the slide (top or bottom), sample, coverslip (top or bottom), or stage. For example, where a sample is placed on a slide in a microscope field of view, by knowing the slide thickness and distance of the slide relative to the objective, the focal plane can be set below or within the slide or sample. After acquiring a first image at the particular position, the focal plane is moved along the z-axis (i.e., reverse focused) away from the slide, and a second image is acquired at a z-position located above the focal plane at which the first image was acquired. Subsequent images can then be acquired by additional reverse focusing steps, or through one or more forward focusing steps (i.e., moving towards the position of the focal plane at which the first image was acquired). Accordingly, by setting the focal plane for the first image from within or below the slide and acquiring a first image, and reverse focusing to acquire a second image, focusing problems associated with dirt or dust on the top surface of a coverslip, for example, which typifies automated focusing systems, can be minimized or avoided.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.